Superagonistic anti-CD28 Antibody Research Articles

A Comparison of Different Immune Activation Strategies to Reverse HIV-1 Latency.

Resting CD4+ T cells are the best characterized component of the latent reservoir. Activation of these CD4+ T cells is needed to optimize transcription and viral replication, and this strategy has been used to measure the inducible reservoir. There are several methods that can be used to activate CD4+ T cells, and in this study, we compared 3 different strategies: the combination of the lectin phytohaemagglutinin (PHA) and irradiated allogeneic feeders, a combination of PHA and a superagonistic anti-CD28 antibody, and the combination of the protein kinase C agonist phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin. We show that each strategy induces a different pattern of expression of activation markers on CD4+ T cells. However, the different activation strategies induced similar frequencies of latently infected CD4+ T cells from people living with HIV on suppressive antiretroviral therapy regimens to produce replication-competent virus. Furthermore, the frequency of infectious units per million induced by each regimen was positively correlated with the copies of intact proviral DNA per million CD4+ T cells. Our results suggest that no single pattern of activation marker expression is most associated with latency reversal and demonstrate that different immune activation strategies reverse latency in a low frequency of CD4+ T cells that harbor intact proviral DNA.

Induction of T regulatory cells by the superagonistic anti-CD28 antibody D665 leads to decreased pathogenic IgG autoantibodies against desmoglein 3 in a HLA-transgenic mouse model of pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune disease of the skin and mucous membranes. Its pathogenesis is based on IgG autoantibodies that target the desmosomal cadherins, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1) and induce intra-epidermal loss of adhesion. Although the PV pathogenesis is well-understood, therapeutic options are still limited to immunosuppressive drugs, particularly corticosteroids, which are associated with significant side effects. Dsg3-reactive T regulatory cells (Treg) have been previously identified in PV and healthy carriers of PV-associated HLA class II alleles. Ex vivo, Dsg3-specific Treg cells down-regulated the activation of pathogenic Dsg3-specific T-helper (Th) 2 cells. In this study, in a HLA-DRB1*04:02 transgenic mouse model of PV, peripheral Treg cells were modulated by the use of Treg-depleting or expanding monoclonal antibodies, respectively. Our findings show that, in vivo, although not statistically significant, Treg cells exert a clear down-regulatory effect on the Dsg3-driven T-cell response and, accordingly, the formation of Dsg3-specific IgG antibodies. These observations confirm the powerful immune regulatory functions of Treg cells and identify Treg cells as potential therapeutic modulators in PV.

CD28 superagonist-mediated boost of regulatory T cells increases thrombo-inflammation and ischemic neurodegeneration during the acute phase of experimental stroke.

While the detrimental role of non-regulatory T cells in ischemic stroke is meanwhile unequivocally recognized, there are controversies about the properties of regulatory T cells (Treg). The aim of this study was to elucidate the role of Treg by applying superagonistic anti-CD28 antibody expansion of Treg. Stroke outcome, thrombus formation, and brain-infiltrating cells were determined on day 1 after transient middle cerebral artery occlusion. Antibody-mediated expansion of Treg enhanced stroke size and worsened functional outcome. Mechanistically, Treg increased thrombus formation in the cerebral microvasculature. These findings confirm that Treg promote thrombo-inflammatory lesion growth during the acute stage of ischemic stroke.

Abstract 211: Regulatory T cells improve healing after myocardial infarction

Background: The proinflammatory activation of innate immunity by myocardial ischemic injury has been recognized for long time. Our recent data have indicated that activation of CD4+ T cells, presumably by auto-antigen recognition, is a prerequisite for formation of a stable scar and prevention from left ventricular dilation after experimental myocardial infarction in mice. We here hypothesized that regulatory CD4+CD25+Foxp3+CD4+ T cells might improve left ventricular wound healing and prevent from adverse remodeling after myocardial infarction. Results: Experimental myocardial infarction in mice induced the proliferation and activation of CD4+CD25+Foxp3+ regulatory T cells, as demonstrated by intracellular expression of the Ikaros family transcription factor Helios, in heart draining lymph nodes. Pretreatment of mice with an anti-CD25 antibody before myocardial infarction efficiently depleted CD4+CD25+Foxp3+ regulatory T cells and increased mortality after myocardial infarction as compared to mice treated with an isotype-matched control antibody of irrelevant specificity, i.e., 25% survival in anti-CD25 treated mice vs. 55,9% survival in control antibody treated animals. Therapeutic activation of regulatory CD4+CD25+Foxp3+ T cells by a superagonistic anti-CD28 antibody (CD28-SA) applied at day 2 after myocardial infarction prevented, compared to mice treated with an isotype-matched control antibody, from left ventricular rupture and resulted in improved survival (47.1% survival in the control group vs. 76.6% survival in the CD28-SA treated group). CD28-SA treatment lead to expansion of CD4+CD25+Foxp3+ T-cells in the peripheral blood and increased their frequency in the infarcted myocardium. This was associated with increased expression of several molecules known to facilitate wound healing by promoting the formation of a stable scar such as osteopontin and coagulation factor XIII. Conclusion: CD4+CD25+Foxp3+ regulatory T-cells are a prerequisite for proper myocardial wound healing and can be therapeutically activated to improve outcome after experimental myocardial infarction.

Requirements for Growth and IL-10 Expression of Highly Purified Human T Regulatory Cells

Human regulatory T cells (T(R)) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human T(R) cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9.3) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting T(R) cell proliferation, and under these conditions the proliferative capacity of T(R) cells was comparable to conventional CD4 lymphocytes. Blocking TGF-β activity abrogated IL-10 expression from these cells, while addition of TGF-β resulted in IL-10 production. These data demonstrate that highly purified populations of T(R) cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of T(R) cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-β is critical to enable human T(R) cells to express IL-10.

Foxp3+ Regulatory T Cells Control Persistence of Viral CNS Infection

We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4+ CD25+ Foxp3+ Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8+ T cells predominantly recognising the H-2Db-presented viral hemagglutinin epitope MV-H22–30 (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8+ effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS.

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice

To use mice with chronic hyperproliferative skin inflammation as psoriasis models, their thorough phenotypic and functional characterization is indispensable. Mice with keratin 5 promoter-controlled overexpression of latent human Transforming Growth Factor (TGF)beta1 within the basal epidermis (K5.TGF beta 1 mice) show a psoriasiform phenotype, but the underlying pathogenic mechanisms are not entirely clear. To elucidate the contribution of T lymphocytes to the pathogenesis in K5.TGF beta 1 mice, we used three complementary approaches: first, peripheral T cells were eradicated via systemic treatment with CD3- or CD4-depleting antibodies. However, this elimination did not alleviate the chronic inflammatory disorder. Second, bone marrow transplantation from transgenic mice into wildtype recipients and vice versa resulted in the expected reconstitution of both adaptive and innate immune system but had little effect on the cutaneous phenotype both in wildtype and transgenic chimeras. Third, based on the hypothesis that the disease course could be modulated by regulatory T cells (Tregs), we expanded Tregs in vivo using a superagonistic anti-CD28 antibody. While this treatment achieved a threefold increase in Foxp3-expressing Tregs, there was little, if any, effect on the chronic skin inflammation. We conclude from our findings that T cells play little, if any, role in the skin lesions of K5.TGF beta 1 mice.

The calm after the cytokine storm: lessons from the TGN1412 trial

In March 2006, a phase I study of the superagonistic anti-CD28 antibody TGN1412 caused a massive cytokine storm and multiorgan failure in six healthy human volunteers. Such a profound impact on the immune system was not predicted by preclinical animal studies. In a study from this issue of the JCI, Müller et al. treated rats with the superagonistic anti-CD28 antibody JJ316 and found that it rapidly induced a marked T cell lymphopenia by trapping T cells in the spleen and lymph nodes (see the related article beginning on page 1405). This dramatic redistribution of T cells simulated the profound T cell lymphopenia observed in human recipients of TGN1412. In contrast, JJ316 treatment in the rats did not reproduce the massive cytokine storm observed following TGN1412 administration to the human volunteers. These results point to similarities as well as differences between rodents and humans in the immunological effects of superagonistic anti-CD28 antibody treatment and raise further questions about how best to design preclinical studies that can better predict the risks of novel immunotherapeutics in humans.

Ras/PI3Kinase/cofilin‐independent activation of human CD45RA+ and CD45RO+ T cells by superagonistic CD28 stimulation

T cell activation requires costimulation of TCR/CD3 plus accessory receptors (e.g. CD28). A hallmark of costimulation is the dynamic reorganization of the actin cytoskeleton, important for receptor polarization in the immunological synapse. The classical model of T cell costimulation was challenged by the detection of superagonistic anti-CD28 antibodies. These induce T cell proliferation and--as demonstrated here--production of IFN-gamma, CD25 and CD69 even in the absence of TCR/CD3 coligation. Here, we analyzed whether superagonistic CD28 stimulation induces costimulatory signaling events. Costimulation leads to phosphorylation of the actin-bundling protein L-plastin and dephosphorylation of the actin-reorganizing protein cofilin. Cofilin binds to F-actin only in its dephosphorylated form. Binding of cofilin to F-actin leads to depolymerization or severing of F-actin. The latter ends up in smaller F-actin fragments, which can be elongated at the free barbed ends. This results in enhanced actin polymerization. Dephosphorylation of cofilin requires activation of Ras and PI3Kinase. Interestingly, superagonistic CD28 stimulation activates human peripheral blood T cells independently of Ras and PI3Kinase. Accordingly, it does not lead to cofilin dephosphorylation and receptor polarization. Likewise, L-plastin is not phosphorylated. Thus, superagonistic CD28 stimulation does not mimic costimulation. Instead, it leads to a Ras/PI3Kinase/cofilin-independent state of "unpolarized T cell activation".

Superagonistic anti-CD28 antibodies: potent activators of regulatory T cells for the therapy of autoimmune diseases

This paper reviews the existing evidence regarding the use of superagonistic anti-CD28 antibodies (CD28 superagonists) for therapeutic manipulation of regulatory T cells (Treg cells). The molecular properties of superagonistic anti-CD28...

Superagonistic Anti-CD28 Antibody TGN1412 as a Potential Immunotherapeutic for the Treatment of B Cell Chronic Lymphocytic Leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is characterised by an accumulation of malignant B cells, and impaired humoral and cellular immune responses. Evasion strategies of leukemic cells appear to involve down-regulation of co-stimulatory molecules as well as increased resistance to apoptosis. Here we provide data supporting a novel concept to treat B-CLL with a humanized, superagonistic monoclonal antibody specific for CD28 (TGN1412). Superagonistic anti-CD28 antibodies have been shown to activate human T cells in vitro without requirement for engagement of the T cell antigen receptor (Luhder et al., J. Exp. Med. 2003. 197(8):955–66). Indicative of their activation, TGN1412-triggered T cells from healthy donors upregulate, among other activation markers, CD40L, that has been reported to promote anti-leukemic effects when ectopically expressed on B-CLL cells (Wierda et al., Blood . 2000. 96 (9): 2917–2924). In this report, the responses of PBMCs from B-CLL patients to soluble TGN1412 were examined. We show that in a dose-dependent fashion, polyclonal T cell activation was induced by TGN1412 including proliferation, cytokine production and induction of activation markers such as CD25, CD71, CD134 (Ox40), CTLA-4 (CD152) and CD154 (CD40L). Significantly, modulation of malignant B-CLL cells was also observed. MHC class II molecules (HLA-DR), CD95 and the co-stimulatory molecules CD80 and CD86, but not the proliferation marker Ki-67, were strongly up-regulated upon TGN1412 stimulation. These data suggested that improved antigen-presenting functions of B-CLL cells were induced by TGN1412. Accordingly, preliminary data indicate that B-CLL cells isolated from TGN1412 stimulated cultures induced enhanced proliferation of both allogeneic and autologous T cells, and importantly, TGN1412 activated T cells exhibited enhanced CTL-activity against B-CLL cells. In conclusion, our data suggest that TGN1412 induces polyclonal T cell expansion and activation as well as increased APC function of B-CLL cells. They imply that TGN1412 may have future therapeutic benefit for B-CLL patients.